Peanut allergy is a common food allergy that is associated with fatal anaphylactic reactions. Diagnosis is complicated by the presence of sensitized subjects (as assayed by IgE or skin prick test) that can consume peanut with no clinical reaction. Standard treatment for peanut allergy is vigilant avoidance and administration of epinephrine in case of accidental exposure. Recent clinical trials of oral and sublingual immunotherapy have demonstrated efficacy, but there is no established in vitro diagnostic assay to monitor response to therapy. While allergy is an IgE mediated disease, accumulating evidence suggests that allergen specific CD4+ T cells play a significant role in disease development. Significant technical challenges, including low frequencies and the presence of multiple peanut allergens, have limited the study of peanut specific CD4+ T cells in subjects with peanut allergy. The overarching theme of this proposal is establishing a mechanistic correlation between the frequency, phenotype, and molecular signature of peanut specific CD4+ T cells and allergic status, i.e. non-allergic, sensitized but tolerant, clinically allergic, and de-sensitzed or tolerized (following IT). There will be three specific aims. Aim 1 will utilize tetramer assays and CD154 up-regulation assays to characterize peanut specific CD4+ T cells in subjects with different allergic status. Our key objective is to define a T cell frequency threshold that can identify subjects with true peanut allergy. Other objectives include studying T cell responses toward individual peanut allergens based on disease status and association of HLA type with T cell reactivity towards specific peanut allergens. Aim 2 will utilize RNA-seq and Fluidigm microarrays to study the transcriptional signature of peanut specific CD4+ T cells in allergic and non-allergic subjects. Our main objective will be to define a unique transcriptional signature that is associated with allergic phenotypes. The function of some of the novel gene identified in Th2 cells will also be studied. Aim 3 will apply the results and tools obtained in the previous aims to develop a routine diagnostic to measure the efficacy of immunotherapy. We will test the hypothesis that the frequency, phenotype and molecular signature of peanut reactive T cells correlate with the efficacy of immunotherapy treatment by characterizing T cells from subjects in an ITN oral peanut immunotherapy trial.